A central interest in developmental, reproductive, and stem biology is how common precursor cells acquire instruction to differentiate into specialized cell types in various organs. Understanding how cell fates are determined not only satisfies the curiosity on how tissues form, but also has a great implication in controlling and manipulating the differentiation program for tissue regeneration and therapeutical purposes. The main goal of this proposal is to understand how fetal and adult Leydig cell lineages, the cell types responsible for masculinization and fertility of the male, are established. Fetal and adult Leydig celis are two distinct androgen-producing celis that appear at different developmental stages and exhibit unique morphological and molecular characteristics. Defects in the establishment of fetal and adult Leydig cell populations or their ability to produce hormones have a profound impact on differentiation of male reproductive tract, spermatogenesis, and fertility. It is therefore essential to understand how these two Leydig celi populations arise and their differentiation is regulated. Our preliminary results suggest that fetal and adult Leydig cells originate from a common precursor in fetal life and the hedgehog (Hh) signaling pathway is responsible for the separation of these two Leydig celi lineages from the common precursor population. We therefore propose to 1) investigate the effects of ectopic activation of the Hh in the differentiation of fetal and adult Leydig cells, 2) isolate the putative Leydig cell precursors and examine their ability to differentiate into adult Leydig cells, and 3) examine the contribution of Gli1-positive interstitial cells to adult Leydig cells. This application will not only provide an insight into the biological basis of cell fate determination in testes, but will also have clinical relevance by identifying processes susceptible to disorders of male sexual development.